Unraveling the biology of human pathogens is fundamental toward understanding mechanisms of pathogenesis and identifying genes essential for survival in the host. This application focuses on the protozoan parasite Trypanosoma brucei, which causes devastating diseases in humans and animals in sub-Saharan Africa. There are no vaccines, and therapeutic drugs have serious side effects and decreasing efficacy. T. brucei relies on a single insect vector, the tsetse fly for its transmission. At present there are few investigations on the molecular aspects of parasite biology in the tsetse vector and specifically about the signals that promote differentiation to epimastigote forms, migration to the salivary glands or the final differentiation step that results in infectious metacyclic forms. In the last five years there has been a tremendous acceleration in all fields of trypanosome biology due to the completion of the genome sequence and the wide application of RNA interference (RNAi), namely downregulation of gene expression by homologous double-stranded RNA. In addition, exquisitely sensitive, high-throughput sequencing technologies (RNAseq) have become available to analyze gene expression patterns (the transcriptome) both qualitatively and quantitatively. Since our discovery of RNAi in 1998, this pathway has been a focus of our investigations, which have led to the recent finding that RNAi functions both in the nucleus and in the cytoplasm and to the identification of five core RNAi genes. How these RNAi factors functions and interface with one another and what is the biological scope of nuclear RNAi are some of the questions we propose to investigate. These studies have implications for reconstructing RNAi in RNAi-negative trypanosomatids and for improving applications of the RNAi tool. In addition, in collaboration with Dr. Serap Aksoy at Yale, who is an expert in tsetse biology and maintains the only tsetse colony in the Americas, we will examine the transcriptome during the T. brucei developmental cycle in the fly, with the long term goal of identifying genes essential for survival within the insect vector. We believe that the range of expertise contributed by Dr. Aksoy and our laboratory offers a unique opportunity for synergism.